Pursuing dynamics of minimal residual leukemic subclones in relapsed and refractory acute myeloid leukemia during conventional therapy

Abstract Background Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied. Method Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored. Results Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse. Conclusions In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by heterogeneous molecular and cytogenetic profiles. 1,24][5] Although evidence such as clinical, molecular/genetic, morphologic, and immunophenotypic parameters has accumulated for a long time, [6][7][8] elucidating their clinical relevance in terms of AML recurrence and treatment persistence at the subclonal level remains a challenge.
The current treatment for AML patients at an early stage has been standardized as high-intensity, cytotoxic drug combination therapy for the past 40 years. 6,9Initial treatment can induce temporary remission in approximately 60%-80% of younger and 40%-60% of older AML patients, 10 but relapse occurs in most patients.2][13] Thus, understanding the heterogeneous clonal architecture of AML that evolves during hematopoiesis is indispensable for identifying profitable treatment strategies for AML.
Here, we chronologically investigate drug-resistant MR leukemic subclones in AML patients throughout the treatment process.The practical approach employed in this study, utilizing droplet digital polymerase chain reaction (ddPCR) assay combined with multiparameter immunophenotyping, enables us to explore the clonal promiscuity among MR leukemic cells in the bone marrow of R/R AML patients.

| Whole exome sequencing
Whole exome sequencing (WES) was performed on BM aspirates and matched saliva samples from R/R AML patients.Genomic DNA was extracted from the BM aspirates and matched saliva samples, and DNA libraries were constructed using the SureSelect XT Human All Exon Kit (V5) (Agilent Technologies, Santa Clara, CA).Pairedend sequencing was performed using the NovaSeq 6000 System (Illumina Inc., San Diego, CA, USA).

| Droplet digital PCR
For single nucleotide polymorphism (SNP) detection, genomic DNA was extracted from 7 sorted cell fractions of patient samples using a REPLI-g Mini Kit (150,023, QIAGEN, Hilden, Germany).Following the manufacturer's instructions, the concentrations of sorted cells were adjusted to 600 cells/μL in 0.5 μL and reacted overnight for whole genome amplification.To investigate patient specific leukemic clones within the AML BM pool that may contribute to relapse and treatment resistance in patients, we selected six representative leukemic variants demonstrating significant changes during the patient treatment process among the 11 leukemic variants.Total six variantspecific TaqMan probe assays were designed for the ddPCR assay (Table S2).Variant-specific TaqMan probe assays for two representative leukemic variants from each patient were designed for the ddPCR assay (Table S2).Before analyzing samples, assay conditions were optimized by performing serially diluted wild-type DNA loading, and "no template controls" (NTCs) were run in parallel to avoid amplicon contamination issues.Wildtype and altered sequences were detected in the amplified gDNA using a fluorescein/hexachloro (FAM/HEX) twocolor detection ddPCR system (QX200, Bio-Rad).Variant allele frequency (VAF) values for each target were calculated as the percentage (%) of positive droplets divided by the sum of positive and negative droplets (total accepted droplets), indicating the frequency of variant clones.We noted the minimum detection level of the ddPCR assay (at least three dots, VAF >0.1%). 14However, because we sought to observe the eradication and repopulation of MR subclones, all data are presented in our results.

| Detection of leukemic subclones in longitudinally collected patient samples
We first collected three relapsed and refractory (R/R) AML patients (n = 3) who initially received conventional chemotherapy.As conventional chemotherapy, standard 7 + 3 induction was administered to these three patients, and complete remission (CR) was achieved.They received 3 cycles of high-dose ara-C consolidation or allogeneic peripheral blood hematopoietic stem cell transplantation (allo-PBSCT), but relapsed within a year.After reinduction chemotherapy, second remission (CR2) was achieved in three patients.They received additional donor lymphocyte infusion (Patient #1) or allo-PBSCT (Patients #2 and #3) as consolidation therapy, but Patients #1 and #2 showed treatment persistency (Per) after secondary relapse (Rel2), and Patient #3 showed mixed chimerism suspicious of impending relapse (Table 1).
From 3 R/R AML patients, 15 longitudinally collected BM total nucleated cell (TNC) samples were organized (Table S1), and 8 out of 15 organized samples were used for somatic variant screening.Among the 11 screened oncogenic (or likely oncogenic) variants, 6 out of 11 leukemic variants (Patient #1: FLT3 D835E(A>C) , WT1 R375P ; Patient #2: KIT D816Y , ASXL1 A722L ; Patient #3: KIT N822K , FLT3 D835E(A>T) ) showing significant changes during the treatment process were finally selected (Table 2).The frequency of leukemic clones with selected leukemic variants in seven sorted BM cell fractions (four stem/progenitors: HSCs/MPPs, LMPPs, CMPs/MEPs, and GMPs; three mature leukocytes: myeloid cells, T lymphocytes, and B lymphocytes) (Figure S1) was determined using the designed probe assay.Based on the measured variant allele frequency (VAF) values, we examined the clonal distribution of leukemic subclones in the BM of patients with R/R AML.
In the ddPCR analysis of Patient #1, the clones with leukemic variant WT1 R375P , which were not detected by NGS analysis, were initially found at very low frequencies in the HSC/MPP, CMP/MEP, and GMP (0.11, 0.04, and 0.02%, respectively) populations.Despite the initial treatment leading to an overall reduction in mutant clones at CR1, these mutant clones unexpectedly resurfaced and expanded across all seven BM cell populations at Rel1.The WT1 R375P mutant clones in GMP and myeloid cell populations demonstrated treatment persistence after relapse treatment, similar to FLT3 D835E mutant clones.Moreover, these persisting clones repopulated another mature leukocyte population (T cells: 0.34%), even after achieving a second remission (CR2) (Figure 1B; Figure S2A).
In Patient #1, FLT3 D835E(A>C) and WT1 R375P mutant in some cell populations display treatment persistence and dynamic changes in their cellular phenotypes throughout treatment.These leukemic subclones were likely indicative of minimal residual (MR) disease cells in Patient #1 with R/R AML.In Patient #2, in contrast to the NGS analysis, ddPCR analysis showed that the clones with the KIT D816Y leukemic variant resided only in the LMPP and GMP (0.05% and 0.8%, respectively) populations at an extremely low frequency.As observed in Patient #1, these few mutant clones exhibited persistence following initial treatment and repopulated actively across all progenitors as well as mature leukocytes at CR1.Among these, treatmentpersistent clones in progenitor populations tended to show active clonal expansion at Rel1, whereas those in mature leukocytes showed a significant reduction.These persister clones eventually remained only in the HSCs/ MPPs, LMPPs, and GMPs (0.15%, 13.42%, and 1.39%, respectively) after relapse treatment at CR2 (Figure 2A; Figure S2B).The ASXL1 A722L mutant leukemic clone of Patient #2 was absent at initial diagnosis (Dx).However, the mutant clones appeared in CMPs/MEPs and GMPs at CR1 and subsequently in all BM cell populations, except for the B cell population, regardless of the treatment.Similar to the above KIT D816Y mutant clones, the ASXL1 A722L mutant clones showing treatment persistence with repopulating capacity exhibited varied responses to relapse treatment.Although relapse treatment and allo-PBSCT effectively suppressed mutant clones in mature leukocytes (myeloid cells), mutant clones in the progenitor populations persisted and eventually remained in HSCs/MPPs, LMPPs, and GMPs (0.03%, 29.48%, 1.98%, and 0.03%, respectively) (Figure 2B; Figure S2B).
In Patient #2, the persister clones occupying the progenitor populations seemed to show relatively robust clonal expansion and treatment persistence compared to those in mature leukocyte populations.The primitive BM cell populations are likely to carry treatment-persistent MR leukemic subclones securely in R/R AML patients.

| Leukemic subclones can sustain MR disease properties without clonal expansion
In the ddPCR analysis of Patient #3, the KIT N822K mutant was observed at low frequencies in almost all BM cell populations.These mutant clones generally persisted after the initial treatment and subsequently repopulated even into a new population (GMPs: 0.09%) at CR1.These  treatment-persistent leukemic clones at Rel1 exhibited robust expansion in four progenitor and myeloid cell populations, with slight increases and persistence in T cells (0.2% to 1.15%) and B cells (0.04% to 0.05%).Although relapse treatment and allo-PBSCT led to an overall reduction in the number of persistent clones, these clones eventually remained at low frequencies (CR2) (CMPs/MEPs, myeloid, T, and B cells: 0.10, 0.37, 0.29, and 0.28%, respectively) (Figure 3A; Figure S2C).The FLT3 D835E(A>T) mutant clone of Patient #3, detected at 6.37% in NGS analysis, was not observed in the ddPCR analysis of seven BM cell fractions at diagnosis (Dx).However, at first remission (CR1), mutant clones appeared in LMPPs, CMPs/MEPs, GMPs, and T cells (0.03, 0.05, 0.03, and 0.08%) at extremely low frequencies.In contrast to the other persisting clones, these treatmentpersistent clones, which showed no markable expansion (VAFs below 0.5%) at Rel1, were irregularly distributed across the seven populations at extremely low frequencies throughout the treatment process.(Figure 3B; Figure S2C).
Two treatment-persistent clones in Patient #3 showing distinct changes in their VAF values may indicate the coexistence of two independent MR leukemic subclones.In addition, they can sustain MR disease properties throughout the treatment process by residing in various cell types at a low frequency without clonal expansion.

| DISCUSSION
This study continuously monitored minimal residual (MR) disease clones in the bone marrow (BM) of patients with relapsed and refractory (R/R) AML, rather than identifying novel therapeutic targets of LSCs.Using NGS and ddPCR genotyping methods combined with multiparameter immunophenotyping analysis used in previous studies, 14,19,22 the dynamics of AML MRD clones were traced throughout the patient's treatment process.
Using this combined approach, we sought to identify clones bearing leukemic variants within the BM cell population of patients.In three patients, some leukemic clones within the leukemic cell pool exhibited persistent responses to AML treatment.In addition, these treatmentpersistent clones were irregularly distributed in various types of cells, including mature leukocytes, and were greatly expanded in the relapse stages.We were able to identify these treatment-persistent clones as MR leukemic subclones that might be associated with treatment refractoriness and recurrence of AML.However, owing to phenotypic heterogeneity, we were unable to categorize the MR leukemic subclones according to their cellular phenotypes in our three cases.This study showed chronological features of MR leukemic subclones during the treatment process of R/R AML in practical way.To date, multiple studies have revealed that CD34+/ CD38− cells represent the phenotype of pre-existing drug-resistant clones and leukemic stem cells (LSCs) in AML BM. 23,24 Consistent with this, our analysis showed that CD34-expressing MR leukemic subclones of primitive phenotypes (HSCs/MPPs, LMPPs, CMPs/MEPs, and GMPs) actively repopulated other cell populations.MR leukemic subclones with primitive phenotypes appeared to be an initial contributor not only carrying MR disease properties themselves but also transmitting them to other populations with distinct phenotypes.Our results demonstrated that CD34-expressing AML MR clones partially represented a small number of true LSCs that withstood AML treatment and expanded greatly in relapse/treatment persistent stages. 25n this study, we hypothesized that specific clones with leukemic variants showing significant changes after the clinical remission stage might contribute to AML relapse and acquire treatment resistance as MRD-like clones in the BM.Therefore, we investigated two representative leukemic clones in each patient and demonstrated two features of MR subclones: (1) a small number of preexisting MR leukemic subclones and (2) dynamic changes in their phenotypes, regardless of the hierarchical order of normal hematopoiesis.Considering the clonal promiscuity of MR clones that emerge during these treatments, the study of a direct correlation between MR clones and AML relapse and drug resistance is left to our future studies.
By proposing a practical method to track MR leukemic subclones, this study reveals the clonal promiscuity of MR disease in the BM of R/R AML patients.We emphasize that proactive suppression of AML MRD clones is crucial for inhibiting AML onset.

T A B L E 2
Abbreviations: 7 + 3, 7 days of cytarabine arabinoside (ara-C) and 3 days of idarubicin; Allo-PBSCT, allogeneic peripheral blood hematopoietic stem cell transplantation; AML, acute myeloid leukemia; CLAG-M, combination chemotherapy of cladribine, ara-c, G-CSF, and mitoxantrone; F, female; FLAG-I, combination chemotherapy of fludarabine, ara-c, granulocyte colony stimulating factor (G-CSF), and idarubicine; GVHD, graft-versus-host disease; HDAC, high dose of ara-C consolidation; M, male; MSD, matched sibling donor; MUD, matched unrelated donor; OS, overall survival since the initial diagnosis of AML; PFS1, progression-free survival (PFS) since the achievement of complete remission (CR) after the 1st induction treatment to 1st relapse; PFS2, PFS since the achievement of the secondnd CR after re-induction treatment to second relapse.

F I G U R E 1
The distribution of FLT3 D835E and WT1 R375P mutant clones from 7 bone marrow (BM) cell fractions in Patient #1.Cells from longitudinal AML BM samples were sorted by phenotype into 7 cell fractions (4 HSCP populations and 3 mature leukocytes) using flow cytometry.The frequencies of FLT3 D835E and WT1 R375P mutant clones in the sorted cell fractions were determined using ddPCR.Light blue: induction therapy; green: consolidation therapy; red: allogeneic G-CSF-mobilized peripheral blood stem cell transplantation.(A) Frequency of the FLT3 D835E mutant clone (variant allele frequency, VAF) distributed in HSCs/MPPs, LMPPs, CMPs/MEPs, GMPs (left), myeloid cells, T cells, and B cells (right) during treatment.The blue line represents the VAF in the entire BM obtained by the bulk WES.(B) Frequency of the WT1 R375P mutant clone distributed in seven cell fractions during treatment.The red line represents the fraction of mutant clones in the total bone marrow identified by bulk WES.

3 . 3 |
Leukemic subclones with primitive phenotypes are the main carriers of MR disease properties

F
I G U R E 2 KIT D816Y and ASXL1 A722L mutant clones distributed in 7 BM cell fractions of Patient #2.AML BM cells were analyzed and sorted into 7 cell fractions according to their phenotype profiles.Allele frequencies of KIT D816Y and ASXL1 A722L variants, indicating the fractions of mutant clones, were determined using a ddPCR assay.Light blue: induction therapy; green: consolidation therapy; red: allogeneic G-CSF-mobilized peripheral blood stem cell transplantation.(A) Changes in the frequency of the KIT D816Y mutant clone determined by ddPCR in 7 cell fractions during treatment.The blue line represents the VAF in the entire BM obtained by the bulk WES.(B) Changes in the frequency of the ASXL1 A722L mutant clone in 7 cell fractions were determined by ddPCR.The red line represents the fraction of mutant clones in the total bone marrow identified by bulk WES.

F I G U R E 3
Distribution of KIT N822K and FLT3 D835E mutant clones and BM constitution in Patient #3.The BM cells were sorted into 7 cell fractions based on their cellular phenotypes.Variant allele frequencies of mutant cells were determined in genomic DNA obtained from 7 sorted cell fractions using ddPCR.Light blue: induction therapy; green: consolidation therapy; red: allogeneic G-CSF-mobilized peripheral blood stem cell transplantation.(A) Frequency changes in the KIT N822K mutant clone in 7 cell fractions were determined by ddPCR during treatment.The blue line represents the VAF in the entire BM obtained by the bulk WES.(B) Frequency changes in the FLT3 D835E mutant clone in 7 cell fractions were determined by ddPCR.The red line represents the fraction of mutant clones in the total bone marrow identified by bulk WES.